: Abstract: This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-102: Chemist/Biologist collaborations facilitating tool development. The goal of this project is to develop a new set of molecular reagents that deliver chemical probes to specific ion channel complexes functioning in living cells. The development of this novel technology will enable the facile determination of the subunit composition and membrane localization of ion channel complexes in native cells. For this project, we will target the N-linked glycans on ion- conducting and regulatory subunits of two K+ channels found in cardiomyocytes: Kv11 K+ channels and KCNE type I transmembrane peptides. There are three aims of this project. In Aim 1, we will derivatize two peptide toxins such that when they bind to their cognate K+ channel complex they will covalently label the complex with either biotin or a fluorescent probe. The labeling efficiency and specificity of the derivatized peptide toxins will be demonstrated in electrical recordings, biochemical assays and fluorescence imaging experiments. Aims 2 and 3 will demonstrate the utility of the panel of derivatized toxins by identifying the native regulatory KCNE subunits co-assembled with cardiac K+ channels and determining the cell surface localization and turnover of K+ channels in living cardiomyocytes. Ion channels function as macromolecular protein complexes composed of membrane-embedded ion-conducting and regulatory subunits. Complex assembly is vital for proper cellular function, as mutations that prevent co-assembly give rise to neurological, cardiac, muscular and respiratory diseases. This project describes the development of a novel set of reagents to probe the structure, function and cellular localization of healthy and diseased ion channel complexes.